Granulocyte colony stimulating factor (GCSF) is a cytokine produced by macrophages, endothelial cells, monocytes, and fibroblasts. Human GCSF consists of 174 amino acids with an approximate molecular weight of 19.60 kDa. GCSF plays a critical role in the modulation of neutrophil biology. GCSF is required for maintaining an adequate basal neutrophil count, as well as for the generation of an appropriate neutrophilia in response to infectious stimuli. GCSF stimulates the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. It regulates them using janus kinase (JAK)/signal transducer and activator of transcription (STAT) and ras/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signal transduction pathways.
GCSF increases the neutrophil cell division and decreases marrow transit time, leading to an increase in the total amount of neutrophils. Secondary effects of rHu GCSF on neutrophils include attraction and localization to sites of infection, increase in phagocytosis and a decrease in apoptosis. rHu GCSF was approved by FDA for use in chemotherapy induced neutropenia m 1991 (Global Regulatory Affairs, Sanofi US). rHu GCSF is a 175 amino acid non glycosylated protein expressed in E. coli with molecular weight of 18.8 kDa. rHu G-CSF contains an additional N-terminal methionine, which is essential for its expression in E. coli cells.
The expression of recombinant proteins is mainly achieved by using prokaryotic or eukaryotic expression systems. Prokaryotic expression systems offer several advantages including, cost, culture conditions, rapid cell growth, yield and relatively short expression time over eukaryotic expression systems. However the key drawback associated with the prokaryotic expression system is the absence of glycosylation. rHu G-CSF produced using E. coli cells forms inclusion bodies in the form of insoluble protein aggregates.
The development of cost effective and efficient downstream processes is an essential part of the biopharmaceutical manufacturing processes. Process robustness, scalability, reproducibility, and capability of selective removal of product and process related impurities are essential requirements of an industrial biopharmaceutical manufacturing process. Breakthrough discoveries in molecular biology and upstream processes in recent years are responsible for higher recombinant protein titre which has shifted the overall economics of manufacturing processes towards downstream processing. Although this bottleneck can be overcome by scaling up the process, there still exists a physical limit of existing facilities throughput and scalability. With biotechnology companies operating under ever-increasing pressure towards lowering the cost of manufacturing processes, integration of cost effective alternative purification strategy is the need of the hour. This problem clearly defines the need for efficient purification strategies for manufacturing of recombinant proteins. In view of this bioprocess technologists are investigating the role of alternative purification strategies which are capable of providing economically efficient large scale processes. Few alternatives among these are, use of precipitation or selective extraction techniques for the isolation of the product of the interest. Aqueous two phase system (ATPS) is an attractive alternative which offers a solution to the above mentioned problems, by increasing the overall throughput and minimizing the cost of manufacturing the product.
Aqueous two phase systems forms as a result of the incompatibility between two aqueous phases of various polymers or the salt solutions. Albertson in 1955 demonstrated use of polyethylene glycol and phosphate based aqueous two phase system as separation tool for downstream processing of various biological molecules (Albertsson, P. Å. Advances in Protein Chemistry, 1970, 24, 309-341). Till date aqueous two phase system based separation processes has been applied to many biological systems which mainly include purification of proteins, nucleic acids, plant or animal cells, microorganisms etc. Selective isolation of the target protein in one of the phases of aqueous two phase system forms the basis of the purification in ATPS. Process parameters which affect this selectivity in partioning includes type of the polymers, type of the salt, concentration of polymer as well as salt, pH of the system, ionic strength of the system etc. Certain properties of biological molecules affect the selectivity, such as charge, hydrophobicity, molecular weight, conformation etc.
In the recent years there is a paradigm shift in the application of the aqueous two phase system from crude purification technique for cell or microorganism separation to polishing step technique for downstream processing of biologicals. Rosa et al have enlisted the application of aqueous two phase systems for purification of various biopharmaceutical proteins (Rosa et al., Journal of Chromatography, 2010, 1217, 2296-2305; Rosa et al., Journal of Chromatography A, 2007, 1141, 50-60; Rosa et al., Journal of Chromatography A, 2009, 1216, 8741-8749; Rosa et al., Journal of Chromatography A, 2010, 1217, 2296-2305). However in comparison to the existing techniques such as chromatography the recovery and purity values which are obtained using ATPS is still a fact of the concern. For various biopharmaceuticals using aqueous two phase systems the recovery values range from 50-95% with the purity levels ranging from 76-95% (Andrews et al., Journal of Chromatography B: Biomedical Sciences and Applications, 1996, 685, 5-20; Andrews et al., Bioseparation, 1996, 6, 303-313; Azevedo et al., Separation and Purification Technology, 2009, 65, 14-21; Azevedo et al., Journal of Chromatography A, 2008, 1213, 154-161; Azevedo et al., Journal of Biotechnology, 2007, 132, 209-217; Azevedo et al., Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 2009, 877, 50-58; Azevedo et al., Separation and Purification Technology, 2009, 65, 31-39; Ferreira et al., Journal of Chromatography A, 2008, 1195, 94-100).
WO 2010146599 describes the application of aqueous two phase system for the purification of rHu GCSF. The recovery of rHu GCSF from the process employed is 40% to 50%.
Further, isolation of various product related impurities in biopharmaceutical proteins is achieved using various chromatographic techniques. Different chromatographic techniques are based on the differences in physicochemical interaction of the components with the resin matrix. Various commonly used include affinity, ion exchange, and hydrophobic interaction-chromatography. Ion-exchange chromatography (IEC) forms the backbone of most biopharmaceutical drug purification processes as it offers high selectivity for isolation of various process and product related impurities.
In the current era of “biosimilars”, additional challenges have been imposed on chromatography process development for matching the product purity profile to the innovator's molecule. Although ion exchange chromatography offers a solution for selective isolation of various product related impurities, critical drawback associated with conventional ion exchange matrix involves intolerance to the high ionic strength protein solutions, need of the buffer exchange step for lowering ionic strength of the process intermediate samples which in turn increases the cost of the manufacturing.